The assembly of N-linked glycoproteins in the endoplasmic reticulum (ER) can be considered to occur in four stages: l) Synthesis of the long chain lipid, dolichyl phosphate (Dol-P); 2) Stepwise assembly, via cytoplasmic sugar nucleotides, of a lipid-linked oligosaccharide chain oriented toward the lumen; 3) Transfer of this oligosaccharide chain to nascent polypeptide chains; and 4) Folding and formation of disulfide bonds of the newly completed (glyco)protein in the lumen. Given the wide-spread distribution and the diversity of function of glycoproteins, it is important to understand how they are assembled in the normal and diseased state. Because much evidence points to a common mechanism of glycoprotein synthesis in yeast and higher eukaryotes, and because yeast offers great advantages in terms of genetic manipulation, the proposed studies will be carried out in the yeast S. cerevisiae. Specific Aim l involves identification of the enzymatic steps in formation of Dol-P. Despite considerable work in a variety of eukaryotic systems, the steps culminating in formation of Dol-P are not well defined. A novel screen will be used to identify temperature sensitive yeast mutants defective in synthesis of Dol-P and thereby determine the pathway of its synthesis. In addition, genes in this pathway that encode for novel enzymes will be cloned and sequenced. In Specific Aim 2 initial studies will focus on the reactions and topology of oligosaccharyl-PP-Dol synthesis. Although previously developed assays will be used to study the topology of the process, a new in vivo genetic approach will also be taken. Thus, a collection of temperature sensitive strains will be screened for mutations in the oligosaccharyl-PP-Dol pathway. If defects in novel reactions in the pathway are detected, the genes will be cloned, sequenced and characterized. However, a major objective will be to identify mutants in the pathway that are defective in either sugar nucleotide transporters or saccharide-lipid translocators. If such a defect is identified the gene will be cloned, and the role of the gene product in transport or translocation will be studied in vivo and in vitro. Specific Aim 3 entails studies on the role of protein disulfide isomerase (PDI) in protein folding and disulfide bond formation in the ER. A key objective will determine the molecular basis for the finding that PDI expression is essential for yeast viability. First, the possible involvement of the Cys residues in PDI will be tested by mutating them. Second, site-directed mutations will be carried out in an acidic domain of yeast PDI that has homology with a domain in rat PDI that binds peptides, and is postulated to be the site for binding of polypeptide chains. In vivo experiments will be carried out to distinguish between two models for PDI binding: Transient interaction with nascent polypeptides versus formation of a stable complex with a mature ER protein.